Antiviral effect and mechanism of Phillyrin and its reformulated FS21 against influenza

Abstract Background Influenza virus causes significant morbidity and mortality with pandemic threat. Oleaceae Fructus Forsythiae is a medicinal herb. This study aimed to investigate antiviral effect of Phillyrin, a purified bioactive compound from this herb, and its reformulated preparation FS21 against influenza and its mechanism. Methods Madin–Darby Canine Kidney (MDCK) cells were infected by one of six influenza viruses: five influenza A viruses (IAVs: three H1N1 and two H3N2) and one influenza B virus (IBV). Virus‐induced cytopathic effects were observed and recorded under microscope. Viral replication and mRNA transcription were evaluated by quantitative polymerase chain reaction (qPCR) and protein expression by Western blot. Infectious virus production was assessed using TCID50 assay, and IC50 was calculated accordingly. Pretreatment and time‐of‐addition experiments with Phillyrin or FS21 added 1 h before or in early (0–3 h), mid (3–6 h), or late (6–9 h) stages of viral infection were performed to assess their antiviral effects. Mechanistic studies included hemagglutination and neuraminidase inhibition, viral binding and entry, endosomal acidification, and plasmid‐based influenza RNA polymerase activity. Results Phillyrin and FS21 had potent antiviral effects against all six IAV and IBV in a dose‐dependent manner. Mechanistic studies showed that both suppressed influenza viral RNA polymerase with no effect on virus‐mediated hemagglutination inhibition, viral binding or entry, endosomal acidification, or neuraminidase activity. Conclusions Phillyrin and FS21 have broad and potent antiviral effects against influenza viruses with inhibition of viral RNA polymerase as the distinct antiviral mechanism.

Vaccination is the primary public health measure for the prevention of infectious diseases, including influenza and COVID-19. However, the effectiveness of current influenza vaccines is suboptimal. 5 Hence, effective antiviral agents for chemoprophylaxis and treatment of influenza are critically important for controlling this common and highly contagious respiratory viral infection. Three classes of antiviral agents have been approved worldwide for the treatment of influenza including ion channel blockers (the adamantanes: amantadine and rimantadine), NA inhibitors (oseltamivir, zanamivir, and peramivir), and an inhibitor of influenza cap-dependent endonuclease (baloxavir). 6 Because of widespread drug resistance, adamantanes have little clinical utility and are no longer recommended. Clinically important resistance to NA inhibitors and baloxavir has increasingly become a significant concern. 7,8 Favipiravir is a pro-drug that inhibits viral RNA polymerase by targeting to its subunit PB1 after being converted by host enzymes and has been approved for use against emerging influenza viruses resistant to other antivirals in Japan. 9 However, Goldhill and colleagues have reported that a specific mutation in the PB1 subunit confers resistance to this relatively new antiviral agent and, at the same time, a compensatory second mutation can restore influenza viral fitness. 10

| Time-of-addition experiments
After influenza virus infection, studies have shown that synthesis and export of viral genomes from the nucleus level out at 3hpi and release of virus particles occurs at 8hpi. 20 As such, influenza virus life cycle can be divided into early (0-3 h), mid (3-6 h), and late (6-9 h) stages.

To expand our kinetics analyses, time-of-addition experiments with
Phillyrin or FS21 (100 μg/ml) were conducted in these three stages.
Cellular viral replication (M vRNA) and M1 and M2 mRNA transcription were evaluated in the presence or absence of Phillyrin or FS21 at 0-9, 0-3, 3-6, and 6-9 h after virus inoculation. For example, to evaluate 3-to 6-h time period, cells were infected with IAV (A/Victoria/361/2011) in viral inoculum containing no Phillyrin or FS21 at 0 h. Viral inoculum was removed, and cells were cultured in fresh medium for 3 h, and at 3hpi, Phillyrin or FS21 was added. They remained in the culture for 3 h and were removed at 6hpi. Cells were cultured in the fresh medium without virus or antivirals for an additional 3 h, and cell lysates were collected at 9hpi for quantitative polymerase chain reaction (qPCR) and Western blot analyses.
2.5 | qPCR, Western blot, cytotoxicity, hemagglutination inhibition (HI), NA inhibition (NI), flow cytometry, and endosomal acidification Methods for these experiments are described in detail in the Supporting Information.

| Luciferase assay
A plasmid-based reverse genetics system was used to evaluate influenza viral RNA polymerase activity using Bright-GloTM Luciferase Assay System kit (E2610, Promega) as previously described. 20 Cell lysates were then harvested, and luciferase activity was measured using Wallac Victor (Perkin Elmer) according to manufacturer's instructions.

| Statistical analysis
Experiment data were analyzed with SPSS 17.0 software. Results were presented as means ± standard deviations (SDs). Comparisons between means of two groups were made using an unpaired, twotailed Student's t test. Comparisons between means more than two groups were analyzed using one-way analysis of variance (ANOVA).

| Phillyrin and FS21 reduced influenza virusinduced cytopathic effect (CPE)
MDCK cells were pretreated with Phillyrin or FS21 and infected with one of the six influenza viruses as described above. Direct microscopic observation at 48hpi showed that treatment with Phillyrin ( Figure 1  3.3 | Phillyrin and FS21 had no effect on virus HAmediated HI, virus binding and entry to, endosomal acidification, or viral release from host cells  Taken together, these results indicate that neither Phillyrin nor FS21 suppresses influenza virus binding to, entry of, or release from the host cells. In addition, Phillyrin or FS21 has no effect on endosomal acidification.  I G U R E 3 Phillyrin ("Phi") and FS21 suppressed infectious virus production. Madin-Darby Canine Kidney (MDCK) cells were pretreated with Phillyrin or FS21 and infected with an influenza virus the same way as described in Figure 2. Culture supernatants were collected at 48hpi, and viral titers were determined by TCID 50 assay. The graphs represent the mean and standard deviation of % inhibition TCID 50 by the treatment of Phillyrin (A-F) or FS21 (G-L) from three independent experiments. Each graph represents one of the six influenza viruses as indicated.
T A B L E 1 Fifty percent inhibition concentration (IC50) against influenza viruses based on TCID 50 assay a .  They became weaker when Phillyrin or FS21 was added in a later stage (i.e., 6-to 9-or 3-to 6-h period) but remained significant, except for the suppression of M1 and M2 mRNA transcription by Phillyrin that was no longer statistically significant ( Figure 4C,D). Consistent with these results, Phillyrin and FS21 potently suppressed the expression of viral protein PA, NP, and M1, and such suppression reached almost completion when it was present in the entire 0-to 9-h or early 0-to 3-h periods ( Figure 4H,I, respectively).
These results indicate that Phillyrin and FS21 suppress influenza virus replication as well as viral gene transcription and protein expression efficiently when included throughout the time of infection and with early treatment leading to greater effects than later treatment.

| Phillyrin and FS21 suppressed viral RNA polymerase activity
The effects of Phillirin and FS21 on viral RNA replication suggested that the treatments might target the influenza viral RNA polymerase.  Labdane diterpenoids against IAV H1N1 strain. 25,26 As pointed out earlier, data presented in Figure 4C,D suggest such a possibility.